Tetracycline fermentation



June 4, 1963 Filed Feb. 12, 1962 REFLEGTANGE 8 Sheets-Sheet 1 FlG.-l REFLECTANCE CURVE 0F WHOLE HARVEST MASH OBTAINED WITH 2 AUREOFACIENS STRAIN T-l35 IN CORN STEEP MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 IDA 27 NICHOLAS DEDUOK June 4, 1963 Filed Feb. 12, 1962 REFLECTANCE J. A. GROWICH, JR., ETAL 3,092,556

TETRACYCLINE FERMENTATION 8 Sheets-Sheet 2 F lG.-2 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH g AUREOFACIENS STRAIN T-l40 RN STEEP MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 'm,u= 32 AR AT 430 m l8 INVENTORS JOHN ANDREW GROWICH, JR.

NICHO AS D UCK M )1 I ATTO Y June 4, 1963 J. A. GROWICH, JR., ETAL 3,092,556

TETRACYCLINE FERMENTATION Filed Feb. 12, 1962 8 Sheets-Sheet 5 FIG.-3

REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WlTHi AUREOFACIENS STRAIN T-I43 IN CORN STEEP MEDIJM 7 REFLECTANCE WAVELENGTH IMILLIMICRONS) AR AT 420 TIL LL 59 INVENTORS AR AT 430 TQM -9 JOHN ANDREW GROWICH, JR.

NICHOLAS DE UGK BY ATTO NEY June 4, 1963 J. A. GROWICH, JR., ETAL 3,092,555

TETRACYCLINE FERMENTATION Filed Feb- 12, 1962 8 Sheets-Sheet 4 F lG.-4

REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH i AUREOFACIENS STRAIN T-2 25 4 IN CORN STEEP MEDIUM 7 REFLECTANCE WAVELE NGT'H (Ml LLI M ICRO NS) AR AT 420 'mA 72 INVENTORS AR AT 430 (U LL, 3 JOHN ANDREWGROWICHJR.

NICHOLAS DUCK June 4, 1963 J. A. GROWICH, JR., ETAL 3,092,556

TETRACYCLINE FERMENTATION Filed Feb. 12, 1962 8 Sheets-Sheet 5 FlG.-5 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH QAUREOFACIENS STRAIN 5-2308 IN CO STEEP MEDIUM REFLECTANCE WAVELENGTH (MILLIMICRONS) AR AT 420 m 63 INVENTORS AR AT 430 j I2 7 JOHN ANDREW GROWICH, JR.

NICHOLAS DUCK I ATTOREY REFLECTANCE June 1963 .1. A. GROWICH, JR., ETAL 3,092,556

TETRACYCLINE FERMENTATION Filed Feb. 12, 1962 8 Sheets-Sheet 6 F lG.-6 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN 9-2589 IN CORN STEEP MEDIUM 400 5 0 WAVELENGTH (MILLI MICRONS) AR AT 420 m u. 94 INVENTORS AR AT 430 To 36 JOHN ANDREW GROWICH,JR.

ICHOLS D UCK BY ATTOR EY June 4, 1963 Filed Feb. 12, 1962 REFLECTANCE 8 Sheets-Sheet 7 FlG.-7 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH AUREOFACIENS STRAIN 8-2589 IN SYNTHETIC MEDIUM WAVELENGTH (MILLIMICRONS) AR AT 420 'ITjL 8 AR AT 430 TY/A 87 INVENTORS JOHN ANDREW GROW|CH,JR.

NICHOLAS D UCK ATTOR June 4, 1963 J. A. GROWICH, JR., ETAL 3,092,556

TETRACYCLINE FERMENTATION Filed Feb. 12, 1962 8 Sheets-Sheet 8 FlG.-8 REFLECTANCE CURVE OF WHOLE HARVEST MASH OBTAINED WITH iAUREOFACIENS STRAIN 8-2589 IN DRIED WHEY MEDIUM REFLECTANCE WAVELENGTH (MILLIMIGRONS) AR AT 420 TIL/LL I29 INVENTORS AR AT 430 TtjJ. 66 JOHN ANDREW GROWICH,JR

NICHOLAS DED 0K OR BY United States Patent 3,692,556 TETRACYCHNE FEPMENTATION John Andrew Growich, .lr., New (Iity, N.Y., and Nicholas Deduek, Rahway, N.J., assignors to American Cyanamid Company, New Yorir, N.Y., a corporation of Maine Filed Feb. 12, 1962, Scr. No. 172,394 5 (Iiaims. (-22. 195-40) This invention relates to the production of tetracycline by fermentation and, more particularly, is concerned with certain novel mutant strains of Streptomyces aureofaczens which possess the property of producing tetracycline to the exclusion of chlortetracycline irrespective of the chloride ion content of the fermentation medium.

It has been known for some time that microorganisms of the species Streptomyces aureofaciens which produce tetracycline also simultaneously produce chlortetracycline. The concomitant production of chlortetracycline is objectionable when tetracycline is the principal product sought to be produced. Generally, while specification standards permit small quantities of chlortetracycline to be present in specification quality tetracycline, the presence of any sizable quantities of chlortetracycline is objectionable. Moreover, the presence of these two antibiotics in any sizable amounts in the fermentation mash involves diflicult problems of separation in the refining or extraction procedures. It is possible, of course, to extract the antibiotics from the fermentation mash and by selective refining procedures to eflect a separation of the antibiotics. However, the refining procedures for eflecting separation of the antibiotics are not without some diflicultyand they will usually involve some loss in total antibiotic potency. Moreover, chlortetracycline, which in those instances where tetracycline is the principal product of the fermentation, may be considered a contaminant and is customarily discarded since it is usually not present in suflicient quantity to warrant the expense of a separate purification procedure to bring it up to specification standards. Heretofore, chloride ion deprivation or the use of chlorination inhibitors have been means available for suppressing the formation of chlortetracycline.

The present invention is based'upon the discovery that certain novel mutant strains of Streptomyces aureofaciens produce tetracycline, to the total exclusion of chlortetracycline, by fermentation. These novel mutant strains of Streptomyces aureofaciens are, in essence, chloride ion ignoring strains in that they produce tetracycline exclusively regardless of the concentration of chloride ion in the medium.

The novel mutant strains of the present invention are strains of the species S. aureofaciens since they are all direct descendants of the chlortetracycline-producing S. aureofaciens A-377 soil isolate described in US. Patent No. 2,482,055 to Duggar, and deposited at the Northern Regional Research Laboratories, Peoria, Illinois, and indexed as NRRL-2209. Derivation of these novel mutant strains of S. aureofaciens from the original A-377 strain involved treatment of the A377 strain with mutagenic agents including ultraviolet irradiation, nicotine, and nitrogen mustard. Typical mutant strains of S. aureofaciens which possess the unique property of ignoring chloride ion have been designated by us as T-135, T-l40, T-143, T-225, 8-2308, and 8-2589. Viable cultures representative of these novel mutant strains have been deposited with the American Type Culture Collection (ATCC) in Washington, D.C., and have been assigned the following accession numbers.

In general, the novel mutant strains of the present invention are characteristic of the species S. 'z zrzre of c cieizs but diifer from previously described strains of S. aureofaciens, not only in pigmentation, but also in the colors of the whole harvest mashes obtained therewith. The colors of the whole harvest mashes obtained by fermentation with the novel mutant strains of the present in vention all exhibit a value of AR at 420 m which is greater than the yaiue' of AR at 430 mu.

FIGURES 1 through 8 of the drawings are spectrophotomet'ric reflectance curves of one-centimeter .glass cells filled with the whole harvest mashes obtained with the novel mutant strains of S. z'ziiibfdcins of the present invention. The spectrdphotometric reflectance curve of a material constitutes a permanent record that does not require the maintenance of a sample. Furthermore, the units in which the curve is expressed are understood and accepted in every civilized country. In FIGURES 1 through 8, the wavelength of light in millimicrons is plotted as the abscissa against the reflectance as the ordinate. The wavelength of light has been adopted internationally as the fundamental standard of length to which all other standards of length are referred. These 'spectrophotometric reflectance curves were determined with a standard spectrophotometer using magnesium carbonate as a reference.

The letter R denotes the reflectance value at some particular wavelength, for example, R is the reflectance value at a wavelength of 500 III/1.. The symbol AR denotes the vertical distance between the reflectance curve (when both percent reflectance and wavelength are plotted on linear scales) and a straight line drawn through the reflectance curve intercepts at 400 mu and 550 mp.- This graphical determination of the values of AR at 420 my. and at 430 m may be readily carried out, and in every case the novel mutant strains of the present invention impart a color to the whole harvest mashes such that AR at 420mm is greater than AR at 430 mg.

The mathematical determination of the values ofAR at 420 Inn and at 430 mu may also be accomplished by means of the following equations:

AR at (R550) 130(R4 150(R4 ALR at mp.=30(R )12O(R )150(R wherein k k R4 and R are the reflectance values at wavelengths at 400 111,14, 420 m 430 mg, and 550 11111;, respectively. This mathematical determination is not dependent upon the scale used for either percent reflectance or wavelength.

A whole harvest mash is the untreated mash obtained after the fermentation has proceeded to the point where biosynthesis of the primary product has stopped for all practical purposes. Generally, the antibiotic potency of the fermentation mash ceases to rise appreciably after the fermentation has proceeded for from about toabout hours.

To illustrate the visual color variations among the novel mutant strains of S. aureofaciens which produce tetracycline exclusively, these strains'were grown on 'cornsteep agar and the following observations were made.

COLOR OBSERVATIONSi s. AUREOF'ACIENS: A134 CORN- STEEP AGAR: SIX DAY INCUBATION AT 26.5 C.

Strain Single colonies Mass growth Dark luggage tan Maple. Orange rust Do. Oak brown.. Light brown. Dark luggage tan Yellow maple.

Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

COLOR OBSERVATIONS: S. AUREOFACIENS: APG CORN- STEEP AGAR: SIX DAY INCUBATION AT 26.5 C.

Strain Single colonies Mass growth Luggage tan Light spice brown. Orange rust Maple. Dark luggage tan" D0. Luggage tan D0.

1 Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

FORMULATION OF APG AGAR Same as AP4 agar, except cornsteep level is 6 grams per liter.

The novel mutant strains of S. awreofaciens of the present invention which produce tetracycline exclusively possess the same general characteristics as do the strains which produce both ohlortetracycline and tetracycline, and differ among themselves in the same general manner that the tetracycline-producing and chlortetracycline-producing strains difier from each other as has been described in a number of scientific papers which havebeen published. The data appearing below will serve to further distinguish the novel mutant strains of S. aureofaciens of the present invention from previously known strains of S. aureofacz'ens such as the original A-377 strain available as NRRL-2209.

The novel Streptomyces aureofaciens mutant strains were differentiated from S. aureofaciens A-377 (NM 2209) by observation of growth characteristics on various media incubated at 26.5" C. a I (1) GLYCEROL ASPARAGINE BEEF EXTRAUL AGAR Glycerol percent..- 1.0 L-asparagine fl 0.05 Beef extr n... 0.2 KHePO4 (10.... 0.05 Bacto ne'nr do 1.5 Distilled water, 1 s fln 100.0 pH adjustment with 50% KOH 7.0 Post sterilization n1 7.2

Strepio'myces aureofacz'ens Strain T-135 Strain T-l40 Growth-; Good, hyaline; aplgmen- Good, hyaline; apigmentons to topaz. tons to topaz. Aerial hyphae." Slight becoming moder- Slight becoming moderate; white. ate; white; Sporulation None None. Difiusible pig- -do Do.

ment. Reverse Hyaline; topaz becoming Hyaline; topaz becoming oak brown. oak brown.

Strain T443 Strain LP-225 Growth Good, hyaline; dark lug- Excellent.

gage tan 7 Aerial hyphae Sparse to fair; white be- Abundant to profuse; coming silver grey. white becoming light fawn 1 to beaver. Sporulation Fair Profuse. Difiusizble pig- Light amber Yellow. 7

men Reverse Eveline; deep brown Dark brown mahogany.

Strain A-377 Growth. Good Aerlalhyphae.-- Slight to fair; white to light gray. Sporulation Fair Difiusible pig- Light yellow ment. W 7 Reverse Yellow tOJightorangeyellow.

Colors according to the Color Harmon Container Corporation of America.

y Manual, Third Edition,

(2) DEXTRIN CZAPEK-DOX AGAR Dex-trin percent. 1.0 NaNOa o 0.2 KeHPOr do 0.1 MgSO4-7H2O do-.." 0.05 Km do 0.05 FESO4-7H2O do"-.. 0.001 Bacto n ar do 1.5 Distilled water, q.s 'd0 100.0 Post sterilization on 7.2

Streptomycin aureofaciens Strain l Strain T- Growth Slight, thin, semitrans- Slight, thin, semitranslucent; white. lucent; white. Aerialhyphaa Slight; white None. Sporulation None Do. Difrusible plgdo Do.

ment. Reverse White, translucent White, translucent.

Strain T-143 Strain T-225 Growth Slight, thin semitranslu- Slight, thin, semi-opaque cent; white. white. Aerialhyphae. Slight; white Slight, white. Sporulation N None. Difiusible pig- Do.

ment. Reverse White; translucent White; translucent.

Strain A-377 Growth Good Aerial hyphae.-- Abundant, mouse gray to lead gray; waterwhite surface globules. Stimulation... Profuse Diffusible pig- Trace; pale Yellow ment. Reverse Apigmentous; pink trace" 1 Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

(3) A94 CORNSTEEP AGAR Cornsteep percent 0.4 Sucrose 7 do 1.0 MgSOr-THeO do 0.025 KH2PO4 do 0.2 (NH4)2HPO4 d 0.2 Bacto agar do 2.0 Tap water, (1 e do 100.0 Post sterilization pH do 6.5

M Streptomyces aureofaciens Strain l135 Strain T-MO Growth"; Moderate Moderate. Aeriel hyphae Scarce becoming abun- Moderate; beaver 1 to dent; beaver. chocolate. Sporulation Moderate to abundant-.- Moderate. Soluble pigment Orange-brown Orange-brown. Reverse Light brown to chocolate Chestnut brown to deep brown. brown.

Strain T-143 Strain T-225 Growth Abundant Abundant. Aerial hyphae Abundant; fawn 1 to bea- Profuse; chocolate ver. Sporulatiom. Moderate to abundant Profuse. Soluble pigment. Light orange-brown Light orange-brown. Reverse Deep brown to dark Dark rose brown to brown. ebony brown.

Strain A-877 Growth Excellent Aerial hypha Abundant; fawn Sporulation Profuse Soluble pigment. Light yellow to amber. Reverse Light tan r 1 Colors accordin Container Corporation of America.v

g to the Color Harmony Manual, Third Edition,

(4) APG CORNSTEEP AGAR (6) OTHER AGAR MEDIA Cornsteep rams-.. 6 Strpto'mycee aureo facien s Sucrose do 10 Medium MgSO4-7H2O "do" 0.25 KI'IEPO-i do 2 Strain T-135 Strain T-140 (NH4)2HPO4 d 2 Bacto agar do 20 p Tap water q.s ml 1 000 Nutrient agar. Poor to fair growth; Poor growth; white to 1 Post sterilization pH '65 white to pale yellow. pale yellow. No aerial No aerial hyphae. hyphae, V Reverse: Refirersez white to pale whiteto pale yellow. ye ow. 1o Glucose Aspara- Good growth; hyaline, Good growth; hyaline, Sneptomyces dw gine Meat exapigmentous to pale apigmentous to pale M y tract Agar. oiiangeJR No aerial hyfiranlge Slight aerial 1) ae. everse: apigyp ae ormation; StramT-135 Stram T440, mentous to pale orange. white. Reverse: apig- W k G d th 1 Gmeintoustov fi laleorange. a smans o0 grow sem o0 groi semiq Abundant Abundant agar. opaque; white to pale opaque; white to pale Aerial hyphae Moderate to abundant; Moderate to abundant; orange} Aerial Granger becoming fawn to mlst bmwnbeaver to fawnphae: sparse'be coming orange with age. Aerial Sporulat1on Moderate to abundantm" Profuse. b nda twhite to hyphae: sparse becom- Soluble pigment. Orange to orange-br0wn Orange to orange-brown. fawn 's m abundant,- white to Reverse Chestnut brown to deep Chestnut brown to deep Sparse becoming abm} beaver, s m

brownbrowndant. Reverse: dark sparse becoming proluggageb tan to Idarllli fuse. Regerse: be ilge to spice rown. 1g copper rown. race Stmm T443 strain T-225 amber soluble pigment. of lightt amber soluble pigmen Purple milk Pale yellow growth col- Pale yellow growth colf Abundant Abundant lar. Partial peptonizalar. Partial peptoui- Aerial hyphae." Slight to moderate; beige 1 Profuse; chocolate tionDH 7, Zation, pH 7 Q 9 mist brown- Potato slants Profuse, moist, nodulat- Profuse, moist, nodulated P P Fall Profuseed growth; chestnut growth; light spice Soluble Dlgmellt W fl brown 1 to dark spice brown. Barn red solu- Reverse Chocolate brown Ebony brown. brown Rust brown ble pigment Slight soluble pigment. :vhitef aerial hyphae Strain A-377 9 V 1 Colors according to the Color Harmony Manual, Third Edition, Con- Gmwth Excellent talner Corporation of America. 7 Aerial hyphae Moderate to abundant;

, fawn Streptomycex aureofacz'ene Sporulat1on Profuse Medium Soluble pigment. Orange-brown Reverse Orange to orange-yellow... strain T443 strain T425 10 1 according to the Color Harmony Manual, Third Edition Nutrient agar.-. Poor growth: white to Poortofair growth: white Container Corporation of America. fi orangeyenowj to golden brown} N0 0 aerial hyphae. aerial hyphae. Re- Reverse: whitetolight verse: translucent, orange-ye1low. white to adobe brown.

No soluble pigment. Glucose aspara- Good growth: hyaline, Abundant growth. .Prog'in'e meat e'x'-' pale yellow to oi-ange. fuse aerial hyphae; 40 tract agar. Aerial hyphae: slight, beaver. Sporulation: white. Reverse: hyprofuse. Reverse: dark aline; pale yellow to eggplant. Very light %opper brown to deep amber soluble pigment. rown. (5) Q4 CORNSTEEP AGA'R Waksnians Good growth; light tan 'A'bii'nda'nt growth. agar. to topar. Aerial hy- Abundant aerial hy- Cornsteep ra 9 5 phae: fair to abundant, phae; taupe brown 1 to Sucrose do 10 4 beige brown to fawn. chocolate. Reverse: MgSOr-THeO do 0.25 Sporulation: poor beebony. Light amber (NHOzHPCh 2 coming profuse, Resoluble pigment. Crude agar 30 verse: dark luggagetan Water, s ml 1,000 to tiers brown. llngbliit Post sterilization H 6.5 g g f g e Potato slants Profuse, moist, nodu- Profuse, moist, smooth lated growth; beaver nodulated growth; to light spice brown. orange rust to oak Sirep'iomycer aureofaciens Dense white aerial hybrown becoming chocophae at isolated foei. late with age. Orange Rust brown soluble brown soluble pigment. Strain T-l35 Strain T-140 pigment.

Purple milk. Pale yellow 1 growth 001- Heavy pale yellow 1 lar. Little significant growth collar. Partial Growth Abundant Profuse. pH change or apparent pcptonization, pH 6.78. Aerial hyphae.-. Alundant; fawn 1 to Profuse; fawn 1 to beaver. peptonization, pH 6.7. N o soluble pigment.

eaver. Sorulation. Abundant Abundant. Soluble pigment Deep orange-brown Deep orange-brown. ggg gg gggggg g g i gggg Harmony Manual Thlrd Eamon Reverse Copper brown to deep Copper brown to deep 1 brown brown 0 Streptomyces aureofaciens Strain 'r-143 Strain 'r-225' Medium Strain A 377 Growth Profuse Profuse. Aerialhyp'hae--. Prtofuse; beige to mist Profuse; chocolate. Nutnent agar gg ggggfi fi gi gggfi' gfigz rown Glucose asparagine Good growth. Aerlal hyphae white becomlng 9 Profuse f m b meat extract agar, increasingly gray with increase in spore forma- Soiuble pigment Deep orange-browm. Deep am er. on Trace, yellow, orange Soluble pigment Reverse Deep brown to chocolate Ebony brow-n. Rev'erSe: light yellowto pinkmmnge. brown Waksruans agar--. Good growth. Aerial hyphae fair becoming Strain A 377 abundant: white to taupe brown. Light yellow soluble pigment. Reverse: camel to Excellent; pale yellow Potato slants Piii t s s ir i i t smootll nodulated growthlight f g g g dark brown" um brown-yellow to beige to cedar. No soluble stlript sn' 0211 55515255"- pigment- Pur 1e milk Slight yellow to pale yellow growth collar. Reverse Orange to p Little significant pH change or apparent peptonization in 14 days. pH 6.45.

1 Colors according to the Color Harmony Manual, Third Edition, Container Corporation of America.

(7) MICROSCOPIC OBSERVATIONS Streptomyaes aureojacims Medium Strain T-135 Strain T-l40 Mycelium Spores Mycelium Spores A1 4 corn- Flexuous, spheroidal Flexuous, spheroidal steep agar. oontinuto ovoidal. continuto ovoidal.

ous, Diam. 0.8 ous, Diam. 0.8 branched. to 1.5; branched. to 1.5 Diam. 0.8 Diam. 0.8 to 1.0; to 1.0 Q4 agar Flexuous, spheroidal Flsxuous, spheroidal continutoovoidal. continuto ovoidal. ous, Diem. 1.0 ous., Dlam. 1.0 branched. to 1.2;. branched. to 1.2;. Diam. 0.8 Diem. 0.8 to 1.0 1. to 1.0a. Waksman's Flexuous, spheroidal Flexuous, spheroidal agar. continuto ovoidal. continuto ovoldal.

ous, Diarn. 1.2 cus, Diam. 1.0 branched. to 1.5. branched. to 1.2;. Diem. 0.8 Diem. 0.8 to 1.0 to 1.0

Streptomyces aureofaciens Medium Strain T-143 Strain T-225 Mycelium Spores Mycelium Spores Glycerol Flexuous, Spheroldal Flexuous, spheroidal asparagine continuto ovoidal. continuto ovoidal. meat ous, Diem. 1.0 ous, Diam. 1.2 extract branched. to 1.2 V branched. to 1.5;. agar. Diem. 0.8 Dlam. 0.8

to 1.0,. CO 1.0;. A1 4 corn- Flexuous, spheroidal Flexuous, Spheroidal steep agar. continutoovoidal. continuto ovoldal.

ous,' Dlam. 0.8 one, Diam. 1.0 branched. to 1.5 branched to 1.2;. Diam. 0.8 Diam. 0.8 to 1.2 to 1.0 11.

Streptomyces aureofaciem Medium Strain A-377 Mycelium Spores Glycerol asparagine Flexuous, continuous, Spheroldal to ovoidal. meat extract agar. ilzarailnghed. Diem. 0.8 Diem. 1.0 to 1.5 1. o p. AP4. cornsteep agar Flexuous, continuous, spheroidal to ovoidal.

grainzhed. Diem. 1. Diam. 1.2 to 1.51:.

o p. Y

The conditions of the fermentation are generally the same as for the presently known methods of producing chlortetr-acycline by fermentation. That is, the fermentation medium contains the usual nutrients and mineral substances. Suitable nutrient substances include starch, dextrose, cane sugar, glucose, molasses, soybean meal, milk solids, yeast, meat extracts, peptone, urea, cornsteep liquor, distillers solubles, fish meal and other con ventional substances. The inorganic salts include such things as calcium carbonate, ammonium sulfate, ammonium chloride, and salts of the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation such .as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculum, sterilization, inoculation and the like are conventional and may be similar to those for the production of chlortetracycline shown in U.S. Patent No. 2,482,055 to Duggar.

The recovery of the tetracycline from the fermentation liquor is conventional and need not be described as numerous methods for recovering tetracycline from fermentation, liquors have already been published.

The invention will be described in greater detail in conjunction with the following specific examples.

8 EXAMPLE 1 Inoculnm Preparation of S. aureofaciens Strain T- Spores of S. aureofaciens strain T-l35 were washed from a streaked agar slant with sterile distilled water to form a suspension containing (BO-8 0x10 spores/ml. A 0.33-m1. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of medium prepared according to the following formulation:

Sucrose grams" 30 Ammonium sulfatedo 2 Calcium carbonate do 7 Cornsteep ml l6.5 Tap water, qs ml 1,000

Chlommine-T Test for Tetracycline-Chlortetracycline Difierentiation (Stock solution A was preparedby dissolving 100 grams of Chloramine-T in 900 ml. of 0.1 M Na PO (38 grams of Na PO -12H O per 1,000 ml. of distilled water). A working solution B was prepared by mixing ml. of stock solution A with 820 of an aqueous solution of ethylenediaminetetraacetic acid sodium salt (3 grams per 1,000 ml. of distilled water). In performing the actual test, 0.5 ml. of the fermentation mash to be tested is mixed in a test tube with 2.0 ml. of working solution B. The tube is shaken intermittently for a period varying from 30 to 60 minutes. At the termination of this period of shaking, the test tube is examined for color. Mashes containing high concentrations of tetracycline turn wine red (a positive reaction), while ma shes containing high concentrations of chlortetracycline develop a deep amber color. This Chloramine-T test is a further means of differentiating the novel mutant strains of the present invention from previously known strains of S. aureofaciens. r 7 EXAMPLE 3 Silver Nitrate Test for Chloride Ion To a l0-ml. portion of whole fermentation mash, there is added 10 of 0.2 M HNO and 0.5 gram of a chloride fiee, diatomaceous earth filter aid. The combined materials are mixed thoroughly and then filtered. 'Ihe filtrate thus obtained is re-filtered as often as necessary to obtain a clear filtrate. A 2.-ml. portion of 0.01 M AgNO is next added to a 2-ml. aliquot of the clear filtrate in a test tube. The mixture is observed for a period of 1 minute. The formation of AgCl, evidenced either as turbidity or as a White precipitate, indicates the presence of free unbound chloride ion in the whole mash samples.

EXAMPLE 4 Tetracycline Production in Shaker Flask Fermentations A. FULL CHLORIDE MEDIUM A full chloride fermentation medium for tetracycline production was prepared as follows:

Tap water, q s ml 1,000

B. CHLORIDE-LIMITED MEDIUM A chloride-limited fermentation medium for tetracycline production was prepared as follows:

Five 25-ml. aliquots of th media were placed in ten 250-ml. Erlenmeyer flasks, sealed in each case with a cotton plug, and sterilized in an autoclave for minutes at a pressure of 15 pounds per square inch. After sterilization, each pair of -ml. aliquots were variously inoculated with 1.0 ml. of inocula prepared as in Example 1. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, at pH 67, on a rotary shaker operating at 180 revolutions per minute.

Under these conditions, the results set forth in Table I below were obtained.

TABLE I Assays Chlora- Silver Strain No. Medium mine-T nitrate Chlortetra- Tetratest 1 test 2 cycline, cyeline, meg/ml. meg/ml.

T-135 A 3 50 2, 460 B 50 2,510 T-140 A 50 2,210 B 50 2,200 T443 A 50 3,820 B 50 3,470 T-225 A 50 3,890 B 50 3,090 8-2308 A 50 6, 580. B 50 6, 520

1 Chlorarnine'l test as per Example 2.

2 Silver nitrate test as per Example 3.

3 Thechlortetracycline assaymethod is not sensitive topless than 50 meg/ml. However, the cultures showed no ehlortetracycline by paper strip chromatography.

EXAMPLES Tetracycline Production in Shaker Flask Fermentations- Efiect of Ammonium Chloride Concentration Fermentation medium A of Example 4 was prepared with levels of 0.0, 0.1, 0.2, 0.5, and 1.5 g./l. of NH CI and a constant 5.0 g./l. level of (Ni-19 80 Fermentation was carried out as described in Example 4, with the results set forth in Table II below.

1 Silver nitrate test as per Example 3.

2 The chlortetracyeline assay method is not sensitive to less than 50 meg/ml. However, the cultures showed no chlortetracychne by paper strip chromatography.

10 EXAMPLE-6 Response of Tetracycline-Producing Strains t0 Ultravi0let.Light The novel mutant strains of the present invention may be readily differentiatedfrom previously known chlortetracyclineaproducing strains of S. aureojaciens (as well as from established strains producing primarily other known ,tetracyolines such as 6-demethyltetracycline, 7-chloro-6- demethyltetracycline, and 7chloro-5a(11a)-dehydrotetracycline) by comparison of the fluorescence produced under ultraviolet light of longer wave length, primamily at 3660 A. A medium which lends itselfparticularly well to growth and fluorescence observations is Q, sporulation agar, prepared as follows:

-MgSO -7H O lgramsn 0.25 KH PO do 0.4 (NH HPO do 0.4 Sucrose do 15 Staleys Nutrient #22 do 9 Crude agar do 30 Tap water, q s ml 1000 to form a suspension containing 6080 10 spores/ml.

A 0.1 ml. portion of this suspension was used to inoculate a portion of the Q sp'or'ulation agar slants. A fter inoculation, the Q agar slants were incubated at 26.527.5 C. Sporulation was generally complete after 14 days. The sameprocedure was followed in the case of all the other microorganisms tested which are listed in Table III below.

When observed under ultraviolet light (3660 A.) during the 0- l4 days incubation period, vegetative growth of tetracycline-producing strains became brilliant goldenorange while the butt became increasingly brilliant orange-yellow to yellow. During this phase of growth, chlortetracycline-producing strains may also exhibit a slight amount of fluorescence of a similar type. Simultaneously, the upper portion of the agar slant, where the medium thickness is minimal, fluoresces gree-yellow for tetracycline-producing strains and golden-yellow for chlortetracycline-producing strains. Further, as the slants mature, the tetracycline-producing strains retain the yellow fluorescent response in the agar butt to ultraviolet light at 3660 A. whereas the chlortetracycline-producing strains become pale blue.

A fermentation medium for tetracycline production was prepared according to the following formulation:

The fermentation procedures were carried out according to the directions :given in Example 4, using the micro- 1 1 organisms listed in Table III below, with the results set forth in Table III below.

1 The chlortetraoycline assay method is not sensitive to less than 50 meg/ml. However, the cultures showed no chlortetracycline by paper strip chromatography.

EXAMPLE 7 Characterization of T etracycZine-Proa'ucing Strains of S. Aureofaciens Hy Spectrophotometric Reflectance of the Whole Harvest Matches in Cdrnsteep Medium Spores of S. aureofaciens strain T-135 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80 16 spores/ml. A 0.33 ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of 'a medium prepared according to the formulation disclosed in Example 1. Subsequent preparation of inocula of the novel mutant strains of S. aureofaciens employed followed the method of Example 1.

A fermentation medium of the following composition was prepared:

(-NH SO grams 5.5-7.0 CaCO 7-10 N-H Cl do 1.5-2.0 MnSO (70% tech. grade) do 0.08-0.15 Cornsteep liquor do -30 Cottonseed meal do 0-5 Starch do 45-55 Corn flour do 14.5 Lard oil ml 20-35 Tap water, q.s ml 1000 After sterilization of this medium in an autoclave for 20 minutes at a pressure of 15 pounds per square inch, ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at 180 revolutions per minute. The same procedure was followed in the case of the other microorganisms employed which are listed in Table IV below.

Assays for tetracycline were performed according to the standard Hiscox method while chlortetracycline assays were obtained by a fluorimetric technique. The results are set forth in Table IV below. Reflectance curves were obtained for the whole harvest mashes by the use of a General Electric Co. type spectrophotometer, and are reproduced in FIGURES 1 through 6 of the drawings. The samples were run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 m to 700 me was scanned. The values of AR at 420 mo and at 430 mg were determined and are set forth in Table IV below.

7 TABLE IV Assays AR at S. aureofaciens Strain N o. Chlortet-ra- Tetracycycline, cline, perpercent by 7 cent by 420 m 430 m weight weight 1 The cultures showed no chlortetraeyclinc by paper strip chromatography.

2 The scales of reflectance and wavelength in Figures 1 through 8 are not linear with the quantities plotted. This is due to the particular plotting mechanism of the spectrophotometer used and in no way effects the calculation of the AR values.

EXAMPLE 8 Characterization of a Tetracycline-Producing Strain of S. aureofaciens by Spectrophorometric Reflectance of the Whole Harvest Mash in Synthetic Medium Spores of S. aureofaciens strain S-2589 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80 .10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of a medium prepared according to the formulation disclosed in Example 1.

A fermentation medium of the following composition was prepared:

nation, consisting of 3 parts of lard oil to 1 part of U.S.P.

grade mineral oil, at the rate of 0.63 ml. per 25 ml. of

medium. After sterilization of this medium in an autoclave for 20 minutes at a pressure of 15 pounds per square inch, 25 ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at revolutions per minute.

The whole harvest mash assayed 8060 micrograms of tetraycyline per ml. according to the standard Hiscox assay method whereas the culture showed no chlortetracycline by paper strip chromatography. A reflectance curve was obtained for the whole harvest mash by the use of a General Electric Co. type spectrophotometer, and is reproduced in FIGURE 7 of the drawings. The sample was run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 m to 700 mg. was scanned. The value of AR at 420 m was 118 whereas the value of AR at 430 mg was 87.

13 EXAMPLE 9 Characterization of a T etracycline-Producing Strain of S. aureofaciens by Spectmphotometric Reflectance of the Whole Harvest Mash in Dried Whey Medium Spores of S. aareofaciens strain S2589 were washed from an agar slant with sterile distilled water to form a suspension containing 6080 10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an eight-inch shaker tube containing 8 ml. of a medium prepared according to the formulation disclosed in EX- ample 1.

A fermentation medium of the following composition was prepared:

Dried Whey grams 15.5 Cornfiour do 42.7 Cornstarch do 30.8 CaCo do 11.5 (biHgJgSO d NH Cl do 2.0 MnSO (70% technical grade) do 0.140 CoCl -6H O do 0.005 Lactic acid do 0.650 Tap water, qs mil 1,000

To this medium was added 0.75 ml. of lard oil per 25 ml. of medium. After sterilization of this medium in an autoclave for 20 minutes at a pressure of 15 pounds per square inch, 25 ml. aliquots in 250-ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the inoculum prepared as described above. The fermentation was carried out at 28 C. from 0 to 18 hours and then at 24 C. from 18 to 142 hours, on a rotary shaker operating at 180 revolutions per minute.

The whole harvest mash assayed 6070 micrograms of tetracycline per ml. according to the standard HiscoX assay method whereas the culture showed no chlortetracycline by paper strip chromatography. A reflectance curve Was obtained for the whole harvest mash by the use of a General Electric Co. type spectrophotometer, and is reproduced in FIGURE 8 of the drawings. The sample was run in a one-centimeter glass cell placed in the reflectance position at the back of the sphere; magnesium carbonate was used as the reference. The specular reflectance of the cell was included and a range of 400 m to 700 mg was scanned. The value of AR at 420 mg. was 129 whereas the value of AR at 430 me was 66.

This application is a continuation-in-part of our co- 1d pending application Serial No. 51,974, filed August 25, 1960, now abandoned.

What is claimed is:

1. The process of producing tetracycline which comprises cultivating a strain of Streptomyces aureofaciens which produces tetracycline exclusively under submerged aerobic conditions in an aqueous nutrient medium containing assimilable surces of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced, said tetracycline-producing strain of Streptomyces aureofaciens being also characterized by its ability to impart to the whole harvest mash a color characterized by a reflectance curve, when plotted linearly, wherein the vertical distance between the reflectance curve and a straight line drawn through the reflectance curve intercepts at 400 m and 550 mp. is greater at 420 m than at 430 mu.

2. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13908 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

3. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13909 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

4. The process of producing tetracycline which cornprises cultivating Streptomyces aureofaciens ATCC No. 13910 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

5. The process of producing tetracycline which comprises cultivating Streptomyces aureofaciens ATCC No. 13911 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts until substantial quantities of tetracycline are produced.

FOREIGN PATENTS Great Britain Dec. 4, 1957 

1. THE PROCESS FOR PRODUCING TETRACYCLINE WHICH COMPRISES CULTIVATING A STRAIN OF STREPTOMYCES AUREOFACIENS WHICH PRODUCED TETRACYCLINE EXCLUSIVELY UNDER SUBMERGED AEROBIC CONDITIONS IN AN AQUEOUS NUTRITENT MEDIUM CONTAINING ASSIMILABLE SURCES OF CARBOHYDRATE, NITROGEN, AND INORGANIC SALTS UNTIL SUBSTANTIAL QUANTITIES OF TETRACYCLINE ARE PRODUCED, SAID TETRACYCLINE-PRODUCING STRAIN OF STREPTOMYCES AUREOFACIENS BEING ALSO CHARACTERIZED BY ITS ABILITY TO IMPART TO THE WHOLE HARVEST MASH A COLOR CHARATERIZED BY A REFLECTANCE CURVE, WHEN PLOTTED LINEARLY, WHEREIN THE VERTICAL DISTANCE BETWEEN THE REFLECTANCE CURVE AND A STRAIGHT LINE DRAWN THROUGH THE REFLECTANCE CURVE INTERCEPTS AT 400 MU AND 550 MU IS GREATER AT 420 MU THAN AT 430 MU. 